Team:Lethbridge/Notebook/Lab Work/April


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April 13/2010

(In the Lab: JV, AS)
Objective: Test Restriction Endonucleases for Activity
Relevant Information:
Endonucleases available

EndonucleaseOptimal Buffer(**)Other Buffers
EcoRVNone2xT(100%); O,G(50-100%)
PstIOrangeR(100%); B,G,T,2xT(50-100%)
DpnITangoB,G(100%): O,R,2xT(50-100%)

(*)Star Activity
(**)Optimal Buffer from Fermentas

Use pUC19 plasmid as test, it has cut sites for EcoRI, PstI, XbaI (unsure about BcuI/SpeI, DpnI but will try anyways), and none for EcoRV
Red Buffer: EcoRI, PstI, Control (No Enzyme)
Tango Buffer: BcuI/SpeI, XbaI, DpnI, Control (No Enzyme)

Methods: Set up Master Mixes:

Red MMper tube (µL)Total (µL)
MilliQ H2013.7555
Red Buffer (10x)27
pUC19 (10pg/µL)27

Tango MMper tube (µL)Total (µL)
MilliQ H2013.7555
Tango Buffer (10x)27
pUC19 (10pg/µL)27

To each tube, add 19.75µL of master mix and 0.25µL of enzyme
Incubated reaction mixes at 37oC (Start:7:00pm; End:7:45pm)
Add 3.3µL of 6x loading dye to each reaction mixture and load 10µL final volume onto a 1% agarose (in TAE) gel.
Add 1µL of 6x loading dye to 1µL of GeneRuler 1kb ladder (at 0.5µg/µL)
Gel loading order as follows:

11kb Ladder
2Tango Control
3DpnI (Tango)
4BcuI/SpeI (Tango)
5XbaI (Tango)
6EcoRI (Red)
7PstI (Red)
8Red Control

Ran gel at 100V for 1 hour

Results: pUC19 plasmid DNA not present at a high enough concentration to visualize by ethidium bromide staining (1kb ladder did stain).

Conclusion: Will have to re-run experiment with DNA that is present at high enough concentrations to visualize by ethidium bromide staining